Spheroid formation and functional restoration of human fetal hepatocytes on poly-amino acid-coated dishes after serial proliferation

Primary human fetal hepatocytes proliferated in monolayer culture up to the 9th passage. During proliferation, the cells changed their morphology from a fibroblast-like shape after inoculation to an epithelia-like polygonal shape after they reached confluence. The proliferation was associated with the loss of ammonia detoxification capacity, which is essential for the function of bioartificial liver. The cells formed spheroids on a poly-glutamic acid- or poly-aspartic acid-coated polystyrene dish that had a negatively charged surface at neutral pH. However, the cells did not form spheroids on a poly-lysine- or poly-arginine-coated dish that had a positively charged surface, which is reportedly suitable to form spheroids for adult hepatocytes. The activity of cytochrome P450 (CYP 1A1, CYP1A2) of the cells in spheroid culture was about twice as high as that of the cells in monolayer culture. The ammonia detoxification activity of the cells was restored in spheroid culture by treatment with 2% dimethylsulfoxide. These results suggest that the conditions for human fetal hepatocytes to form spheroids are different from that for adult hepatocytes, and the use of poly-glutamic acid or poly-aspartic acid coating may improve spheroid culture of proliferative human fetal hepatocytes. This revised version was published online in July 2006 with corrections to the Cover Date.     

表达人肝再生增强因子肝细胞系生物人工肝治疗安全性 的动物实验初步研究

目的:评价表达人肝再生增强因子基因的HepG2细胞系的细胞培养上清及细胞裂解物的小鼠急性毒性和近期致瘤性。方法:SPF级昆明种小鼠18只,随机分为空白对照组、细胞培养上清组、细胞裂解物组,每组小鼠各6只,腹腔分别接种空白培养液、细胞培养上清、细胞裂解物0.5ml。连续14天,每天观察记录动物毒性反应,14d后宰杀小鼠,取血测血生化指标,及观察病理改变。结果:各组小鼠均存活。除对照组1例小鼠,细胞培养上清组1例小鼠,细胞裂解物组2例小鼠次日活动稍减少外,均未见异常反应。血液生化检测ALT、AST、AFP、TBIL无明显异常,且各组间无差别。普通光镜下各组动物肝脏病理切片染色均未见明显异常。结论:目的细胞系细胞培养上清、细胞裂解物对实验用昆明小鼠无明确毒副作用及短期致瘤性,可能提供一种安全的可用于生物人工肝新的细胞来源。     

Packed-bed bioreactors for mammalian cell culture: bioprocess and biomedical applications

This article describes the development history of packed-bed bioreactors (PBRs) used for the culture of mammalian cells. It further reviews the current applications of PBRs and discusses the steps forward in the development of these systems for bioprocess and biomedical applications. The latest generation of PBRs used in bioprocess applications achieve very high cell densities (>10(8) cells ml(-1)) leading to outstandingly high volumetric productivity. However, a major bottleneck of such PBRs is their relatively small volume. The current maximal volume appears to be in the range of 10 to 30 l. A scale-up of more than 10-fold would be necessary for these PBRs to be used in production processes. In biomedical applications, PBRs have proved themselves as compact bioartificial organs, but their metabolic activity declines frequently within 1 to 2 weeks of operation. A main challenge in this field is to develop cell lines that grow consistently to high cell density in vitro and maintain a stable phenotype for a minimum of 1 to 2 months. Achieving this will greatly enhance the usefulness of PBR technology in clinical practice.     

Enhancement of albumin secretion by short-term hypothermic incubation of primary rat hepatocytes

A feasibility of hypothermic incubation of hepatocytes as a means of enhancing liver-specific activity was investigated to obtain preferable hepatocytes for a bioartificial liver (BAL) system. Freshly isolated rat hepatocytes were incubated at hypothermic temperatures from 10 to 33 °C for several days, and subsequently cultured at normothermic temperature of 37 °C to evaluate cell viability and albumin secretion activity. The cell viability was decreased by 3-day hypothermic incubations at 10 and 20 °C, while it was maintained even after 3-day hypothermic incubations between 25 and 33 °C. The activity of albumin secretion gradually decreased with prolonging the period of hypothermic incubation at 25 °C. Enhancement of albumin secretion activity was observed in the hypothermic incubations at 30 and 33 °C. The maximum activation of albumin secretion was obtained when hypothermic incubation was performed for 3 days at 30 °C, where the activity increased to 145% of the original activity. The hypothermic incubation at 30 °C also reduced the required time to be the peak of the activity of albumin secretion in the normothermic culture. It was considered that the hypothermic incubation at 30 °C would be effective as a method for pretreatment of isolated hepatocytes for a BAL system.     

细胞三维培养：组织工程的关键技术突破口

组织工程是有望从根本上解决组织,器官缺损或失能的医学难题的一门新兴边缘学科,组织,器官发育的细胞和分子机制的进一步揭示和体外构建工程组织,器官的细胞培养技术的进步将使组织工程在新的千年成为广泛应用的新的治疗模式。细胞三维培养要成为体外构建工程组织,器官的成熟技术体系需先解决以下问题;（1）细胞;（2）生物材料;（3）培养基;（4）培养装置;（5）异型细胞的共培养;（6）细胞三维培养物血管化。     

Chitosan nanofiber scaffold enhances hepatocyte adhesion and function

To enhance cell attachment and promote liver functions of hepatocytes cultured in bioreactors, a chitosan nanofiber scaffold was designed and prepared via electrospinning. Effects of the scaffold on hepatocyte adhesion, viability and function were then investigated. Data showed that hepatocytes on chitosan nanofiber scaffold exhibited better viability and tighter cell-substrate contact than cells on regular chitosan film. In addition, urea synthesis, albumin secretion and cytochrome P450 activity of hepatocytes on chitosan nanofiber scaffold were all 1.5 to 2 folds higher than the controls. Glycogen synthesis was also increased as compared with the controls. These results suggested the potential application of this chitosan nanofiber scaffold as a suitable substratum for hepatocyte culturing in bioreactors.     

An attempt at decision making in tissue engineering: reactor evaluation using the analytic hierarchy process (AHP)

To develop a method of human-oriented evaluation in tissue engineering and regenerative medicine, we applied the analytic hierarchy process (AHP) in the evaluation of tissue engineering reactors. For evaluating a reactor for a bioartificial liver (BAL) support system, we identified five criteria: safety, scalability, cell growth environment, mimicking native liver functions and handling. Based on these criteria, we evaluated six different types of BAL bioreactors by three panelists. Using the AHP method, we successfully ranked BAL systems for bridge use in liver transplantation in the attempt at decision making based on human-oriented evaluation.     

原位胶原酶循环灌注法分离猪肝细胞

本文建立了原位胶原酶循环灌注分离猪肝细胞方法并与离体两步胶原酶灌注法进行了比较。猪门静脉和下腔静脉分剐插管,先用D-Hanks液灌注,再采用自制的循环灌流装置进行胶原酶循环原位灌注分离猪肝细胞,分离后的肝细胞以5×105/ml培养,观察分离和培养7d的肝细胞产量、活率、蛋白质合成功能、葡萄糖合成功能和LDH含量。同时测定离体组的上述指标。研究结果表明采用原位胶原酶循环灌注法每克肝组织分离获得的肝细胞总量为5.1×107,肝细胞活率98.6％,培养7d肝细胞活率89.5％。肝细胞的蛋白质合成功能在培养7d中保持稳定;葡萄糖合成功能从1d时1.05±0.15nmol/cell下降到3d时0.74±0.09nmol/cell;LDH含量在3d较高。原位胶原酶循环灌注法分离的猪肝细胞总量、活率高于离体法;蛋白质合成功能和葡萄糖合成功能强于离体法。因此,原位胶原酶循环灌注分离猪肝细胞方法可获得大量高活率和良好功能的猪肝细胞。     

The in vivo performance of bioartificial pancreas in bone marrow cavity: A case report of a spontaneous diabetic feline

Recent studies reported that bone marrow cavity offers a widely distributed and well-vascularized microenvironment which is a considerable implantation site for bioartificial pancreas (BAP). In this study, the in vivo performance of BAPs in bone marrow was further demonstrated in a spontaneous diabetes animal. Mouse insulinoma cells encapsulating in agarose gel were enclosed in a calcium phosphate cement chamber to create a BAP. Ten BAPs were implanted into the femur bone marrow cavity of a diabetic feline. The preprandial blood glucose level, 2 h glucose curve, serum C-peptide level and physiological conditions of the recipient were recorded perioperatively. Results showed that the cat still suffered from hyperglycemia postoperatively. However, the physiological conditions of feline were improved with an increase of serum C-peptide level. The peak point of 2 h glucose curve decreased from 400 to 165-290 mg/dl. The efficiency of exogenous insulin extended from 2 to 10-14 h postoperatively which reveals that the implanted BAPs had partial function. This case report revealed that BAPs implanted in the bone marrow cavity for the spontaneous diabetic is effective. The implanted BAPs provided therapeutic benefit despite sustained hyperglycemia. Further study shall be considered to improve the outcomes of BAPs transplantation.